Immunization and vaccination by direct administration of nucleotide sequences encoding an immunogenic protein (called DNA or polynucleotide vaccination) has been described in Patent Application WO-A-90 11092. The protein encoded by the inserted nucleotide sequence is capable of being expressed in the cells and of bringing about the development of an immune response. (See also U.S. Pat. Nos. 5,846,946, 5,620,896, 5,643,578, 5,580,589, 5,589,466, 5,693,622, and 5,703,055; Science, 259:1745-49, 1993; Robinson et al., seminars in IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis. 175(1):91-97, 1997; Norman et al., Vaccine, 15(8):801-803, 1997; Bourne et al., The Journal of Infectious Disease, 173:800-7, 1996; and, note that generally a plasmid for a vaccine or immunological composition can comprise DNA encoding an antigen operatively linked to regulatory sequences which control expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell; for instance, from upstream to downstream, DNA for a promoter, DNA for a eukaryotic leader peptide for secretion, DNA for the antigen, and DNA encoding a terminator.) This application envisages the use of naked DNA as well as of DNA contained in liposomes. Preferably, the DNA is introduced into the muscle. The DNA could also be introduced into the skin, into certain organs or into the blood, making it possible for the injection to be carried out in different ways such as the intradermal route, the transcutaneous route, the intravenous route and the like.
The studies which followed the first descriptions of this technique have demonstrated the benefit of using either the intramuscular route for injecting DNA, or the so-called xe2x80x9cgene gunxe2x80x9d method which consists in propelling metallic microparticles, such as gold microparticles coated with DNA, directly into the superficial cell layer of the skin.
J. B. ULMER et al., Science, Volume 259, 19 March 1993, 1745-1749; G. J. M. COX et al., J. of Virology, Volume 67, No. 9, September 1993, 5664-5667 and Z. Q. XIANG in Virology 199, 132-140 (1994), have described DNA vaccination trials using the intramuscular route.
It has also been widely demonstrated that the intramuscular route gives superior results to the intradermal route but that, in the final analysis, the most promising route is the use of xe2x80x9cgene gunxe2x80x9d because, with this technique, the administered doses are much less than the doses required by the intramuscular route. Reference may be made to F. FYNAN et al., in P.N.A.S. USA Volume 90, 11478-11482, December 1993, WO-A-95/20660.
Likewise, D. TANG et al., (Nature 356, 152-154, Mar. 12, 1992) have shown the absence of immune response after the administration of human growth hormone by the intradermal route with the aid of a hypodermic needle. The authors have, on the other hand, demonstrated the obtaining of an immune response with the aid of the xe2x80x9cgene gunxe2x80x9d technique.
Only E. RAZ et al., (P.N.A.S. USA, Vol. 91, 9519-9523, September 1994) have reported that intradermal administration could induce high antibody titres.
For its part, the xe2x80x9cgene gunxe2x80x9d technique has the disadvantage of being difficult and expensive to use, since it requires the preparation and the use of gold particles coated with DNA and their administration with the aid of a special propellant.
Some authors have therefore developed an alternative technique which envisages the use of an apparatus for liquid jet administration. There may be mentioned P. A. FURTH et al. in Analytical Biochemistry 205, 365-368, 1992, who describe the use of the apparatus called Ped-o-jet, which is an injector used to deliver human vaccines into muscle tissues, for the administration of a DNA vaccine. The authors report that the injector can cause DNA to pass through the skin and reach the muscles, the fatty tissue and the mammary tissue of live animals.
H. L. VAHLSING et al., in Journal of Immunolog Methods 75 (1994) 11-22, describe the use of the apparatus called Med-E-Jet for transcutaneous and intramuscular administration.
There may also be mentioned M. JENKINS et al., in Vaccine 1995, Volume 13, No. 17, 1658-1664, who describe the use of jet vaccination into the muscle.
The bovine respiratory syncytial virus BRSV is present worldwide and can cause severe diseases of the lower respiratory tract in bovines, this disease being similar to that caused by the respiratory syncytial virus HRSV in children. During one study, it was found that more than 95% of 2-year old calves were infected with the BRSV virus (Van der Poel et al., Archives of Virology 1993, 133, 309-321).
The need for a vaccine against the BRSV virus is felt but has not given rise to the development of effective vaccines. The first attempts to vaccinate children have led to the appearance of facilitation of the disease after natural infection, suggesting that the vaccination could be dangerous (Anderson et al., Journal of Infectious Diseases, 1995, 171: 1 to 7). It is known, however, that antibodies against the two major surface glycoproteins, F (fusion protein) and G (attachment protein) could play a role in protection (Kimman and Westenbrink, Archives of Virology, 1990, 112: 1 to 25). Numerous studies have also been carried out on mouse-, dog- and ferret-type animal models. On the other hand, vaccination trials on bovines with the purified F protein have not given a conclusive result since, as in children vaccinated with HRSV, the calves developed neutralizing antibodies and nonneutralizing antibodies which could interfere with the immune response during a subsequent infection with the virus (L. D. Nelson et al., Am. J. Vet. Res., Vol. 53, No. 8, August 1992, p. 1315-1321).
The objective of the present invention is to provide an improvement in the vaccination of bovines with DNA, which makes it possible to ensure a vaccination which is at least as effective as vaccination by the intramuscular route or by the xe2x80x9cgene gunxe2x80x9d technique but which is easier and less expensive to use.
Another objective of the invention is to provide such an improvement leading to increased safety, essentially as regards the vaccination residues present in the tissues.
Another objective of the invention, which relates to the vaccination of animals intended for consumption, is also to ensure safety such that the vaccination has no unfavourable effect on the appearance of the meat.
Another objective of the invention is to provide a means for mass vaccination.
A specific objective of the invention is to provide such a vaccine allowing the protection of bovines against the BRSV virus, the IBR virus, the BVD virus or the PI-3 virus.
The Applicants have found that it is possible to meet these objectives by administering the vaccine by the intradermal route with the aid of a liquid injector without a needle, ensuring, at 5 points, distribution of the vaccine essentially in the epidermis, the dermis and the hypodermis. The trials conducted by the Applicants in the field of vaccination of bovines against the bovine respiratory syncytial virus (Bovine Respiratory Syncytial Virus, BRSV) have made it possible to obtain superior immunization results by this route compared to that obtained by the intramuscular route.
The present invention proposes, for the first time, the vaccination of bovines with polynucleotide vaccines (or DNA vaccines or plasmid vaccines) designed for, and administered by, the intradermal route by means of a liquid jet injector without a needle.
The subject of the present invention is therefore a polynucleotide vaccine formula comprising an intradermally effective quantity of a plasmid combining a DNA sequence encoding an immunogen and a promoter allowing the expression of this immunogen in vivo in the cells of the skin, this vaccine formula being suitable for intradermal administration (the cells of the epidermis, dermis and hypodermis are targeted in particular; the administration is intended in particular to present the expressed antigens to the dendritic Langerhans"" cells of the skin, which cells are localized essentially in the epidermis) with an apparatus for liquid jet intradermal administration, in particular the apparatus called Pigjet (manufactured and distributed by Endoscoptic, Laons, France) or an equivalent apparatus delivering the vaccine through a 5-nozzle head under conditions equivalent to the Pigjet. In general, the vaccine formulae according to the invention are suitable for administration with an apparatus for liquid jet administration having from 1 to 10 nozzles, preferably from 4 to 6, still more preferably from 5 to 6.
This requires a vehicle suited to the intradermal route, such as water, buffer, physiological saline, liposomes, cationic lipids and, in general, a vehicle of low viscosity, especially equivalent or close to that of water, and a dose volume which is useful and effective by this route.
In particular, but not exclusively, with an apparatus having 5 or 6 nozzles, that is to say administering the dose through 5 or 6 openings and in the form of 5 or 6 jets of identical volume, the dose volume may be advantageously between 0.1 ml and 0.9 ml, preferably between 0.2 ml and 0.6 ml, preferably of the order of 0.4 to 0.5 ml.
The vaccine formula will comprise an intradermally effective quantity of plasmid which will be in general from 10 ng to 1 mg, preferably from 100 ng to 500 xcexcg, preferably from 0.5 xcexcg to 50 xcexcg of plasmid.
Typically, the invention seeks to administer the vaccine formula at several points so as to optimize the transfection of cells with the plasmids. This results in a preference for the use of an injection head with several holes. This can also be combined with a multi-application of the apparatus, that is to say with the distribution of the vaccinal dose in more than one application of the apparatus at different sites. In a particularly preferred manner, it will be possible to use an apparatus with 5 or 6 holes in mono-application or in multi-application, preferably in double-application.
A typical case of the invention is a DNA sequence coding for an immunogen of the BRSV virus, and in particular for the G and/or F gene from this virus (for example 391-2 strain: R. Lerch et al., Virology, 1991, 181, 118-131).
Another typical case of the invention is a DNA sequence coding for an immunogen of the infectious bovine rhinotracheitis (IBR) virus or bovine herpesvirus (BHV), in particular for the gB gene and/or the gD gene (for example ST strain: Leung-Tack P. et al., Virology, 1994, 199, 409-421).
Another typical case of the invention is a DNA sequence encoding an immunogen of the mucosal disease virus (BVD), in particular the E2 gene and/or the E1 gene (for example Osloss strain: L. De Moerlooze et al., J. Gen. Virol., 1993, 74, 1433-1438). It is also possible to combine genes from different subtypes of BVD, for example from North America and from Europe (A. Dekker at al., Veterinary Microbiol., 1995, 47, 317-329).
Yet another typical case of the invention is a DNA sequence coding for an immunogen of the type 3 parainfluenza virus (PI-3), in particular for the HN gene and/or the F gene, preferably the HN gene (sequence of the F and HN genes which was deposited by H. Shibuta in 1987, GeneBank sequence accession No.=Y00115).
In case of a combination of 2 genes, for example F and G from BRSV, HN and F from PI-3 or gD and gB from IBR, the corresponding sequences can be inserted into the same plasmid or into different plasmids.
Pathogenic agent gene is understood to mean not only the complete gene but also the various nucleotide sequences, including fragments, which retain the capacity to induce a protective response. The notion of a gene covers the nucleotide sequences equivalent to those described precisely in the examples, that is to say the sequences which are different but which encode the same protein. It also covers the nucleotide sequences of other strains of the pathogen considered, which provide cross-protection or a protection specific for a strain or for a strain group. It also covers the nucleotide sequences which have been modified in order to facilitate the in vivo expression by the host animal but which encode the same protein.
It is of course understood that the invention consists in the adaptation of the prior art DNA vaccines to an intradermal administration by an apparatus for liquid jet administration. While this results in modifications in the vaccine formula and in particular in the viscosity, the quantity of DNA and the dose volume to be administered, it goes without saying that the invention applies, moreover, to any DNA vaccine constructs described in the prior art. Persons skilled in the art will therefore be able to refer to the state of the art in the DNA vaccination field and in particular to the documents discussed above.
More specifically, the transcription units used in the vaccine formulae according to the invention will comprise a strong eukaryotic promoter, such as the hCMV IE promoter; a mammalian viral promoter.
The vaccine formula according to the invention may be packaged in a multidose vial, for example of 10 to 100 doses, fitted to an apparatus for liquid jet intradermal administration, preferably the Pigjet.
The subject of the present invention is also a portable bovine vaccination unit comprising an apparatus for liquid jet administration and a suitable vial comprising several doses of a vaccine formula as described above, the apparatus for administration being designed so as to deliver a dose of vaccine formula intradermally.
Preferably, the apparatus for administration comprises an injection head provided with 1 to 10 nozzles, in particular from 4 to 6, preferably 5 or 6.
The apparatus for administration may have the different characteristics given in the detailed description. The preferred types of apparatus for administration are those which reproduce the administration conditions obtained with the Pigjet apparatus.
The subject of the present invention is also the use of a plasmid combining a DNA sequence encoding an immunogen of a bovine pathogen and a promoter allowing the expression of this type of immunogen, for the preparation of a polynucleotide vaccine formula according to the different procedures described above, suitable for intradermal administration with an apparatus for liquid jet administration.
The subject of the invention is also a method of vaccination in which a polynucleotide vaccine formula, as described above, is administered by the intradermal route with the aid of an apparatus for liquid jet administration. The administration of the vaccine may be done by one or more deliveries of a determined volume of formula. Likewise, it is possible to envisage one or more vaccinations distributed over time.
The method of vaccination according to the invention may take into account the data mentioned above as regards in particular the apparatus for administration to be used.